Human interleukin-3 receptor α subunit

ABSTRACT

PCT No. PCT/US92/03026 Sec. 371 Date Oct. 13, 1993 Sec. 102(e) Date Oct. 13, 1993 PCT Filed Apr. 16, 1992 PCT Pub. No. WO92/18628 PCT Pub. Date Oct. 29, 1992The present invention relates to the isolation and cloning of the  alpha -chain of the human IL-3 receptor, which, when expressed together with the  beta -chain of the human IL-3 receptor, forms a high affinity receptor for human IL-3. The invention further relates to a method for detecting agonists and antagonists of human IL-3 by the use of a cellular host expressing genes for the  alpha - and  beta -chains of the human IL-3 receptor.

The present application is the United States national applicationcorresponding to International Application No. PCT/US 92/03026, filedApr. 16, 1992 and designating the United States, which PCT applicationis in turn a continuation of U.S. application Ser. No. 07/688355, filedApr. 19, 1991, now abandoned, the benefit of which applications areclaimed pursuant to the provisions of 35 U.S.C. 120, 363 and 365 (C).

FIELD OF THE INVENTION

The invention relates generally to the human interleukin-3-receptor(hlL-3-receptor), and more particularly, to the synthesis of a humanIL-3-receptor component and to the use of the receptor component forscreening agonists and antagonists of human IL-3.

BACKGROUND

Circulating blood cells are constantly replaced by newly developedcells. Replacement blood cells are formed in a process termedhematopoiesis which involves the production of at least eight matureblood cell types within two major lineages: (1) the myeloid lineagewhich includes red blood cells (erythrocytes), macrophages (monocytes),eosinophilic granulocytes, megakaryocytes (platelets), neutrophilicgranulocytes, basophilic granulocytes (mast cells); and (2) the lymphoidlineage which includes T lymphocytes, and B lymphocytes (Burgess andNicola, Growth Factors and Stem Cells (Academic Press, New York, 1983)).Much of the control of blood cell formation is mediated by a group ofinteracting glycoproteins termed colony stimulating factors (CSFs),including G-CSF, M-CSF, GM-CSF, and multi-CSF (also known as IL-3).These glycoproteins are so named because of the in vivo and in vitroassays used to detect their presence. Techniques for the clonal cultureof hematopoietic cells in semisolid culture medium have been especiallyimportant in the development of in vitro assays. In such cultures,individual progenitor cells (i.e., cells developmentally committed to aparticular lineage, but still capable of proliferation) are able toproliferate to form a colony of maturing progeny in a manner which isbelieved to be essentially identical to the comparable process in vive.The role of CSFs in hematopoiesis is the subject of many reviews, and isof great interest to clinical investigators who must treat blooddiseases or deficiencies: e.g. Metcalf, The Hemopoietic ColonyStimulating Factors (Elsevier, N.Y., 1984); Clark and Kamen, Science,Vol. 236, pgs. 1229-1237 (1987); Sachs,Science, Vol. 238, pgs. 1374-1379(1987); Dexter et al., eds., Colony Stimulating Factors (Dekker, N.Y.,1990); and Morstyn et al., Cancer Investigation, Vol. 7, pgs. 443-456(1989).

The biological effects of the CSFs are mediated by specific cell surfacereceptors, which may consist of one or more components. Recently,several of these have been cloned and characterized, e.g. Gearing etal., EMBO J., Vol. 8, pgs. 3667-3676 (1989) (low affinity α-chain ofhuman GM-CSF-receptor); Itoh et al., Science, Vol. 247, pgs. 324-327(1990) (low affinity mouse IL-3-receptor); and Hayashida et al., Proc.Natl. Acad. Sci., Vol. 87, pgs. 9655-9659 (1990) (β-chain of humanGM-CSF-receptor). Besides contributing to an understanding of the signaltransduction process, many of these receptors will be useful screeningtools for agonists and antagonists of the natural ligand. In particular,such tools may lead to the development of non-protein agonists andantagonists which would obviate many of the difficulties associated withprotein therapeutics, e.g. intravenous delivery, short serum half-life,and the like.

SUMMARY OF THE INVENTION

The invention is directed to a component of the human IL-3-receptor,referred to herein as the G-chain of the human IL-3-receptor, and tocompositions thereof which bind with high affinity to human IL-3.Specifically such compositions include an α-chain and β-chain of thehuman IL-3-receptor that can operably associate to form a high affinityreceptor for human IL-3. The invention includes allelic and geneticallyengineered variants of the α-chain-receptor, and nucleic acids encodingthe α-chain-receptor and its allelic and genetically engineeredvariants. Preferably, the receptor component of the invention isselected from the group of polypeptides of the open reading framedefined by the amine acid sequence set forth in SEQ. ID. No. 2(immediately preceding the Claims). Although the listed sequenceincludes the intracellular domain of the α-chain of the receptor, it isclear that truncated forms of the sequence which retain theirextracellular and transmembrane domains and their ability of operablyassociate with the β-chain fall within the concept of the invention.

The invention is based in part on the discovery that high affinitybinding of human IL-3 involves the same β-receptor component as highaffinity binding of human GM-CSF. This led to the discovery and cloningof a cDNA clone, designated pDUK-1, which expresses a protein that iscapable of binding to human IL-3 with high affinity when operablyassociated with the β-chain of a human IL-3-receptor (or equivalently ahuman GM-CSF-receptor), such as encoded by the cDNA insert of pKH97deposited with the American Type Culture Collection (ATCC) (Rockville,Md.) under accession number 40847. pDUK-1 has been deposited with theATCC under accession number 75001. The invention includes nucleic acids(i) that are effectively homologous to the cDNA insert of pDUK-1, and(ii) that encode proteins that form high affinity IL-3-receptors inassociation with the β-chain-receptor protein, e.g. as encoded by pKH97.As used herein, `high affinity` in reference to IL-3-receptor bindingmeans that IL-3 binds to the associated α- and β-chains of the receptorwith a binding constant that is at least an order of magnitude less thanthat for binding to either component alone. More preferably. `highaffinity` means that IL-3 binds to the associated α- and β-chains of thereceptor with a binding constant, K_(d), less than 1 nM, and mostpreferably less than 200 pM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the binding of ¹²⁵ I-labeled human IL-3 to COS 7cells transiently co-transfected with KH97 and pDUK-1; and

FIG. 2 is a restriction map of the vector pME 18.

DETAILED DESCRIPTION OF THE INVENTION

I. Obtaining and Expressing cDNAs for the Human IL-3-Receptor β-Chain

The term "effectively homologous" as used herein means that thenucleotide sequence is capable of being detected by a hybridizationprobe derived from a cDNA clone of the invention. The exact numericalmeasure of homology necessary to detect nucleic acids coding for areceptor α-chain depends on several factors including (1) the homologyof the probe to coding sequences associated with the target nucleicacids that encode polypeptides other than the α-chain, (2) thestringency of the hybridization conditions, (3) the use ofsingle-stranded or double-stranded probes, (4) the use of RNA or DNAprobes, (5) the measures taken to reduce nonspecific binding of theprobe, (6) the nature of the method used to label the probe, (7) thefraction of guanosine and cytidine nucleosides in the probe, (8) thedistribution of mismatches between probe and target, (9)the size of theprobe, and the like. Preferably, an effectively homologous nucleic acidsequence is at least seventy percent (70%) homologous to the cDNA of theinvention. More preferably, an effectively homologous nucleic acid is atleast ninety percent (90%) homologous to the cDNA of the invention. Mostparticularly, an effectively homologous nucleic acid sequence is onewhose cDNA can be isolated by a probe based on the nucleic acid sequenceset forth in SEQ. ID. NO. 1 using a standard hybridization protocol withno more than a few false positive signals, e.g. fewer than a hundred.There is an extensive literature that provides guidance in selectingconditions for such hybridizations: e.g. Hames et al., Nucleic AcidHybridization: A Practical Approach (IRL Press, Washington, D.C., 1985);Gray et al., Proc. Natl. Acad. Sci., Vol. 80, pgs. 5842-5846 (1983);Kafatos et al., Nucleic Acids Research, Vol. 7, pgs. 1541-1552 (1979);and Williams, Genetic Engineering, Vol. 1, pgs. 1-59 (1981 ), to name afew. By way of example, the nucleic acid of SEQ. ID. NO. 1 can be usedas a probe in colony hybridization assays as described by Benton andDavis, Science, Vol. 196, pg. 180 (1977). Preferably, low stringencyconditions are employed for the probe employed. (The dissociationtemperature depends upon the probe length.) For example, for a probe ofabout 20-40 bases a typical prehybridization, hybridization, and washprotocol is as follows: (1) prehybridization: incubate nitrocellulosefilters containing the denatured target DNA for 3-4 hours at 55° C. in5× Denhardt's solution, 5× SSPE (20× SSPE consists of 174 g NaCl, 27.6 gNaH₂ PO₄.H₂ O, and 7.4 g EDTA in 800 ml H₂ O adjusted to pH 7.4 with 10NNaOH), 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA, (2)hybridization: incubate filters in prehybridization solution plus probeat 55° C. for 2 hours, (3) wash: three 15 minute washes in 300-500 mlvolumes of 6× SSC and 0.1% SDS at room temperature, followed by a final1-1.5 minute wash in 300-500 ml of 1× SSC and 0.1% SDS at 55° C. Otherequivalent procedures, e.g. employing organic solvents such asformamide, are well known in the art.

Homology as the term is used herein is a measure of similarity betweentwo nucleotide (or amine add) sequences. Homology is expressed as thefraction or percentage of matching bases (or amine acids) after twosequences (possibly of unequal length) have been aligned. The termalignment is used in the sense defined by Sankoff and Kruskal in Chapterone of Time Warps, String Edits, and Macromolecules: The Theory andPractice of Sequence Comparison (Addison-Wesley, Reading, Mass., 1983).Roughly, two sequences are aligned by maximizing the number of matchingbases (or amino adds) between the two sequences with the insertion of aminimal number of "blank" or "null" bases into either sequence to bringabout the maximum overlap. Algorithms are available for computing thehomology of two sequences: e.g. Needleham and Wunsch, J. Mol. Biol.,Vol. 48, pgs. 443-453 (1970); and Sankoff and Kruskal (cited above),pgs. 23-29. Also, commercial services and software packages areavailable for performing such comparisons, e.g. Intelligenetics, Inc.(Mountain View, Calif.); and University of Wisconsin Genetics ComputerGroup (Madison, Wis.).

Probes based on the nucleic acid sequence of the Sequence Listing can besynthesized on commercially available DNA synthesizers, e.g. AppliedBiosystems model 381A, using standard techniques, e.g. Gait,Oligonucleotide Synthesis: A Practical Approach, (IRL Press, WashingtonD.C., 1984). It is preferable that the probe be at least 18-30 baseslong. More preferably, the probe is at least 100-200 bases long. Probesof the invention can be labeled in a variety of ways standard in theart: e.g. radio-active labels, Berent et al., Biotechniques, pgs.208-220 (May/June 1985), Meinkoth et al., Anal. Biochem., Vol. 138, pgs.267-284 (1984), Szostak et al., Meth. Enzymol., Vol. 68, pgs. 419-429(1979), and the like; and non-radioactive labels, Chu et al., DNA. Vol.4, pgs. 327-331 (1985), Jablonski et al., Nucleic Acids Research, Vol.14, pgs. 6115-6128 (1986), and the like.

Hybridization probes can also be used to screen candidate sources ofα-chain mRNA prior to library construction, e.g. by RNA blotting,Maniatis et al., Molecular Cloning: A Laboratory Manual, pgs. 202-203(Cold Spring Harbor Laboratory, N.Y., 1982); or Hames and Higgins, eds.,pgs. 139-143 in Nucleic Adds Hybridization (IRL Press, Washington, D.C.,1985). Sources of mRNA encoding the desired polypeptides include cellpopulations or cell lines that express, or can be induced to express,large numbers of IL-3-receptors on their surfaces, e.g. in excess of3000-5000.

Preferably, the α- and β-chains of the IL-3-receptor am co-transfectedinto a mammalian expression system (i.e. host-expression-vectorcombination). Many reviews are available which provide guidance formaking choices and/or modifications of specific mammalian expressionsystems: e.g. (to name a few) Kucherlapati et al., Critical Reviews inBiochemistry, Vol. 16, Issue 4, pgs. 349-379 (1984), and Banerji et al.,Genetic Engineering, Vol. 5, pgs. 19-31 (1983) review methods fortransfecting and transforming mammalian cells; Reznikoff and Gold, eds.,Maximizing Gene Expression (Butterworths, Boston, 1986) review selectedtopics in gene expression in E. coli, yeast, and mammalian cells; andThilly, Mammalian Cell Technology (Butterworths, Boston, 1986) reviewsmammalian expression systems. Likewise, many reviews are available whichdescribe techniques and conditions for linking and/or manipulatingspecific cDNAs and expression control sequences to create and/or modifyexpression vectors suitable for use with the present invention, e.g.Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, N.Y., 1982); Glover, DNA Cloning: A PracticalApproach, Vol. I and H (IRL Press, Oxford, 1985), and Perbal, APractical Guide to Molecular Cloning (John Wiley & Sons, New York,1984), to name only a few.

Several DNA tumor viruses have been used as vectors for mammalian hosts.Particularly important are the numerous vectors which comprise SV40replication, transcription, and/or translation control sequences coupledto bacterial replication control sequences, e.g. the pcD vectorsdeveloped by Okayama and Berg, disclosed in Mol. Cell Biol., Vol. 2,pgs. 161-170 (1982) and in Mol. Cell Biol., Vol. 3, pgs. 280-289 (1983),both of which are incorporated herein by reference; the SV40 vectorsdisclosed by Hamer in Genetic Engineering, Vol. 2, pgs. 83-100 (1980),and in U.S. Pat. 4,599,308, both of which are incorporated herein byreference; and the vectors additionally containing adenovirus regulatoryelements, disclosed by Kaufman and Sharp in Mol. Cell Biol., Vol. 2,pgs. 1304-1319 (1982), and by Clark et al. in U.S. Pat. 4,675,285, bothof which are incorporated herein by reference. COS7 monkey cells,described by Gluzman, Cell, Vol. 23, pgs. 175-182 (1981) and availablefrom the ATCC (accession no. CRL 1651), are usually the preferred hostsfor the above vectors. SV40-based vectors suitable formammalian-receptor expression have been developed by Aruffo and Seed,Proc. Natl. Acad. Sci., Vol. 84, pgs. 3365-3369 and 8573-8577 (1987).

II. Binding Assays

Binding assays are accomplished by letting a ligand of unknownspecificity or affinity compete with a known amount or concentration oflabeled human IL-3 for receptor-binding sites of a sample of cellstransfected or transformed with pDUK-1, or its equivalent. Preferably,the IL-3 is labeled by radioiodination using standard protocols, e.g.reaction with 1,3,4,6-tetrachloro-3α, 6β-diphenylglycouril described byFraker et al., Biochem. Biophys. Res. Commun., Vol. 80, pgs. 849-857(1978) (and available from Pierce Chemical Co. as lodogen). Generally,the binding assay is conducted as described by Lowenthal et al., J.Immunol., Vol 140, pgs. 456-464 (1988), which is incorporated byreference. Briefly, aliquots of cells are incubated in the presence of¹²⁵ I-labeled human IL-3 in a final volume of 200 μl culture medium inmicrofuge tubes at 4° C. Cell-bound ¹²⁵ I-labeled IL-3 was separatedfrom non-bound ¹²⁵ I-labeled IL-3 by centrifugation through an oilgradient (10,000× G for 2 min). Nonspecific binding is measured in thepresence of a 100-fold excess of partially purified unlabeled humanIL-3.

The following Examples illustrate but do not limit the invention:

EXAMPLES Example I Construction of cDNA library from TF-1 cells andisolation of pDUK-1

Poly(A)⁺ RNA from human TF-1 cells (Kitamura et al., J. Cell Physiol.,Vol. 140, pgs. 323-334 (1989)) cultured in the presence of hlL-3 (5ng/ml) was isolated by the guanidium isothiocyanate method (Chirgwin etal., Biochemistry, Vol. 18, pgs. 5294-5299 (1978)), and was converted todouble-stranded cDNA using oligo(dT) primers. After Bst XI linkers(containing Xba I sites) were ligated to both ends of the cDNAs, thecDNAs were size-fractionated through an agarose gel. cDNAs greater than1.0 kb were digested with Xba I and ligated with Xba I-digested pME 18,an SV40-based mammalian expression vector, diagrammed in FIG. 2, to forma library of about 3×10⁶ independent clones. About 3 μg of miniprep DNAfrom pools of 3×10³ clones was co-transfected with 50 ng of pKH97(carrying a cDNA insert encoding the β-chain of the hGM-CSF-receptor)into COS 7 cells by electroporation (0.4 gap cuvette at 300 volts and300 μF using a Gene Pulser (BioRad, Richmond, Calif.)). The Cos 7 cellswere incubated for 72 hours prior to screening. pDUK-1 was isolated byscreening for cells capable of high affinity binding to ¹²⁵ I-labelledhlL-3. 10 nM ¹²⁵ I-labelled hlL-3 was added to transfected Cos 7 cellsin a Chamber Slide (Labo-Tek), after which cells binding ¹²⁵ I-labelledhlL-3 were identified by microscopic autoradiography.

Example II Binding of hlL-3 to COS 7 cells Co-transfected with pKH97 andpDUK-1

A total of 5 μg of equal amounts of pKH97 and pDUK-1 plasmid DNA wastransfected into semi-confluent COS 7 cells by the DEAE-dextran method.72 hours after transfection, the cells were harvested and analyzed inIL-3 binding assays. Duplicates of 2×10⁵ COS 7 cells in 0.1 ml of RPMI1640 containing 10% fetal calf serum, 2 mM EDTA, 0.02% sodium azide and20 mM Hepes (pH 7.4) were incubated for 3 hours at 4° C. with variousconcentrations of ¹²⁵ I-labeled human IL-3 with or without an excessamount of non-labeled human IL-3. The cell-bound radioactivity wasmeasured by separating the cells from free ligand by centrifugationthrough an oil layer, as described by Schreurs et al., Growth Factors,Vol. 2, pgs. 221-233 (1990). IL-3 was iodinated by a standard protocol,that of Chiba et al., Leukemia, Vol. 4, pgs. 22-36 (1990). Briefly, 5 μgof E. coli-produced human IL-3 was incubated in 30-50 μl of 50 mM sodiumborate buffer (pH 8.0) with 1 mCi of the dried Bolton and Hunter reagentfor 12-16 hours at 4° C. Glycine was added to 2.5 mg/ml to stop thereaction and the iodinated IL-3 was separated from the free Bolton andHunter reagent by a PD-10 column. The iodinated human IL-3 had aspecific radioactivity of (4 to 8)×10⁷ cpm/μg and was stable for abouttwo months in Hepes-buffered Hanks's balanced salt solution containing0.1% gelatin, 0.1% bovine serum albumin, and 0.02% sodium azide.

FIG. 1 shows the receptor-binding data. Open circles correspond to COS 7calls transfected with pKH125 and pKH97. Scatchard analysis (by theLIGAND program, De Lean et al., Mol. Pharmacol., Vol. 21, pgs. 5-16(1982)) of the binding data indicated an equilibrium binding constant,K_(d), of 100 pM.

Example III Co-transfection of pKH97 and pDUK-1 into NlH3T3 Cells

A DNA fragment containing the neomycin-resistance gene, neo, wasinserted into pKH97 downstream of the SRα promoter to form pKH97neo, anda DNA fragment containing the hygromycin-resistance gene, hyg, wasinserted into pDUK-1 downstream of the SRα promoter to form pDUK-1 hyg.NlH3T3 calls were stably transfected with pKH97neo and pDUK-1 hyg by thecalcium-phosphate procedure, described by Chen and Okayama, Mol. CellBiol., Vol. 7, pgs. 2745-2752 (1987), which reference is incorporated byreference. Stable co-transfectants were selected by 1 mg/ml of G418 and1 mg/ml hygromycin. Analysis of the binding of ¹²⁵ I-labelled hlL-3indicated a K_(d) of about 100 pM.

Example IV. Use of Stably Co-transfected NlH3T3 cells to screen for IL-3Antagonists

Aliquots of NlH3T3 cells co-transfected with pKH97neo and pDUK-1 hyg asdescribed above are distributed to wells of microtiter plates in 200 μlof medium containing ¹²⁵ I-labeled human IL-3 at concentrations of 100pM, 500 pM, and 1 nM. 100 μl samples of microbial supernatants free ofcells are added to the transfected NlH3T3 cells at each of the differentconcentrations of ¹²⁵ I-labeled IL-3. After incubation for 3 hours theNlH3T3 cells are harvested and assayed for bound radioactivity. NlH3T3cells with low counts of bound radioactivity correspond to microbialsamples containing candidate antagonists or agonists of human IL-3.

The descriptions of the foregoing embodiments of the invention have beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention tothereby enable others skilled in the art to best utilize the inventionin various embodiments and with various modifications as are suited tothe particular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

Applicants have deposited pKH97 and pDUK-1 with the American TypeCulture Collection, Rockville, Md., USA (ATCC), under accession numbers40847 and 75001, respectively. These deposits were made under conditionsas provided under ATCC's agreement for Culture Deposit for PatentPurposes, which assures that the deposit will be made available to theUS Commissioner of Patents and Trademarks pursuant to 35 USC 122 and 37CFR 1.14, and will be made available to the public upon issue of a U.S.patent, which requires that the deposit be maintained. Availability ofthe deposited plasmids is not to be construed as a license to practicethe invention in contravention of the rights granted under the authorityof any government in accordance with its patent laws.

The Deposits have been modified to satisfy the requirements of theBudapest Treaty on the Deposit of Microorganisms.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES:2                                                   (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1460 bases                                                        (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Encodes Human IL-3-receptor                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCACACGGGAAGATATCAGAAACATCCTAGGATCAGGACACCCCAGATCTTCTCAACTGG60                AACCACGAAGGCTGTTTCTTCCACACAGCACTTTGATCTCCATTTAAGCAGGCACCTCTG120               TCCTGCGTTCCGGAGCTGCGTTCCCGATGGTCCTCCTTTGGCTCACGCTGCTC173                      MetValLeuLeuTrpLeuThrLeuLeu                                                   15                                                                            CTGATCGCCCTGCCCTGTCTCCTGCAAACGAAGGAAGATCCAAACCCA221                           LeuIleAlaLeuProCysLeuLeuGlnThrLysGluAspProAsnPro                              10-515                                                                        CCAATCACGAACCTAAGGATGAAAGCAAAGGCTCAGCAGTTGACCTGG269                           ProIleThrAsnLeuArgMetLysAlaLysAlaGlnGlnLeuThrTrp                              101520                                                                        GACCTTAACAGAAATGTGACCGATATCGAGTGTGTTAAAGATGCCGAC317                           AspLeuAsnArgAsnValThrAspIleGluCysValLysAspAlaAsp                              253035                                                                        TATTCTATGCCGGCAGTGAACAATAGCTATTGCCAGTTTGGAGCAATT365                           TyrSerMetProAlaValAsnAsnSerTyrCysGlnPheGlyAlaIle                              404550                                                                        TCCTTATGTGAAGTGACCAACTACACCGTCCGAGTGGCCAACCCACCA413                           SerLeuCysGluValThrAsnTyrThrValArgValAlaAsnProPro                              55606570                                                                      TTCTCCACGTGGATCCTCTTCCCTGAGAACAGTGGGAAGCCTTGGGCA461                           PheSerThrTrpIleLeuPheProGluAsnSerGlyLysProTrpAla                              758085                                                                        GGTGCGGAGAATCTGACCTGCTGGATTCATGACGTGGATTTCTTGAGC509                           GlyAlaGluAsnLeuThrCysTrpIleHisAspValAspPheLeuSer                              9095100                                                                       TGCAGCTGGGCGGTAGGCCCGGGGGCCCCCGCGGACGTCCAGTACGAC557                           CysSerTrpAlaValGlyProGlyAlaProAlaAspValGlnTyrAsp                              105110115                                                                     CTGTACTTGAACGTTGCCAACAGGCGTCAACAGTACGAGTGTCTTCAC605                           LeuTyrLeuAsnValAlaAsnArgArgGlnGlnTyrGluCysLeuHis                              120125130                                                                     TACAAAACGGATGCTCAGGGAACACGTATCGGGTGTCGTTTCGATGAC653                           TyrLysThrAspAlaGlnGlyThrArgIleGlyCysArgPheAspAsp                              135140145150                                                                  ATCTCTCGACTCTCCAGCGGTTCTCAAAGTTCCCACATCCTGGTGCGG701                           IleSerArgLeuSerSerGlySerGlnSerSerHisIleLeuValArg                              155160165                                                                     GGCAGGAGCGCAGCCTTCGGTATCCCCTGCACAGATAAGTTTGTCGTC749                           GlyArgSerAlaAlaPheGlyIleProCysThrAspLysPheValVal                              170175180                                                                     TTTTCACAGATTGAGATATTAACTCCACCCAACATGACTGCAAAGTGT797                           PheSerGlnIleGluIleLeuThrProProAsnMetThrAlaLysCys                              185190195                                                                     AATAAGACACATTCCTTTATGCACTGGAAAATGAGAAGTCATTTCAAT845                           AsnLysThrHisSerPheMetHisTrpLysMetArgSerHisPheAsn                              200205210                                                                     CGCAAATTTCGCTATGAGCTTCAGATACAAAAGAGAATGCAGCCTGTA893                           ArgLysPheArgTyrGluLeuGlnIleGlnLysArgMetGlnProVal                              215220225230                                                                  ATCACAGAACAGGTCAGAGACAGAACCTCCTTCCAGCTACTCAATCCT941                           IleThrGluGlnValArgAspArgThrSerPheGlnLeuLeuAsnPro                              235240245                                                                     GGAACGTACACAGTACAAATAAGAGCCCGGGAAAGAGTGTATGAATTC989                           GlyThrTyrThrValGlnIleArgAlaArgGluArgValTyrGluPhe                              250255260                                                                     TTGAGCGCCTGGAGCACCCCCCAGCGCTTCGAGTGCGACCAGGAGGAG1037                          LeuSerAlaTrpSerThrProGlnArgPheGluCysAspGlnGluGlu                              265270275                                                                     GGCGCAAACACACGTGCCTGGCGGACGTCGCTGCTGATCGCGCTGGGG1085                          GlyAlaAsnThrArgAlaTrpArgThrSerLeuLeuIleAlaLeuGly                              280285290                                                                     ACGCTGCTGGCCCTGGTCTGTGTCTTCGTGATCTGCAGAAGGTATCTG1133                          ThrLeuLeuAlaLeuValCysValPheValIleCysArgArgTyrLeu                              295300305310                                                                  GTGATGCAGAGACTCTTTCCCCGCATCCCTCACATGAAAGACCCCATC1181                          ValMetGlnArgLeuPheProArgIleProHisMetLysAspProIle                              315320325                                                                     GGTGACAGCTTCCAAAACGACAAGCTGGTGGTCTGGGAGGCGGGCAAA1229                          GlyAspSerPheGlnAsnAspLysLeuValValTrpGluAlaGlyLys                              330335340                                                                     GCCGGCCTGGAGGAGTGTCTGGTGACTGAAGTACAGGTCGTGCAGAAA1277                          AlaGlyLeuGluGluCysLeuValThrGluValGlnValValGlnLys                              345350355                                                                     ACTTGAGACTGGGGTTCAGGGCTTGTGGGGGTCTGCCTCAATCTCCCTGGCCG1330                     Thr                                                                           GGCCAGGCGCCTGCACAGACTGGCTGCTGGACCTGCGCACGCAGCCCAGG1380                        AATGGACATTCCTAACGGGTGGCCTGTGTAATTTCGTTGGGCATGGGAGA1430                        TGCCGAAGCTGCCAGGAAGAAGAACAGAAC1460                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 378 amino acid residues                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Human IL-3- receptor                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValLeuLeuTrpLeuThrLeuLeuLeuIleAlaLeuProCysLeu                              15-10-5                                                                       LeuGlnThrLysGluAspProAsnProProIleThrAsnLeuArgMet                              1510                                                                          LysAlaLysAlaGlnGlnLeuThrTrpAspLeuAsnArgAsnValThr                              152025                                                                        AspIleGluCysValLysAspAlaAspTyrSerMetProAlaValAsn                              30354045                                                                      AsnSerTyrCysGlnPheGlyAlaIleSerLeuCysGluValThrAsn                              505560                                                                        TyrThrValArgValAlaAsnProProPheSerThrTrpIleLeuPhe                              657075                                                                        ProGluAsnSerGlyLysProTrpAlaGlyAlaGluAsnLeuThrCys                              808590                                                                        TrpIleHisAspValAspPheLeuSerCysSerTrpAlaValGlyPro                              95100105                                                                      GlyAlaProAlaAspValGlnTyrAspLeuTyrLeuAsnValAlaAsn                              110115120125                                                                  ArgArgGlnGlnTyrGluCysLeuHisTyrLysThrAspAlaGlnGly                              130135140                                                                     ThrArgIleGlyCysArgPheAspAspIleSerArgLeuSerSerGly                              145150155                                                                     SerGlnSerSerHisIleLeuValArgGlyArgSerAlaAlaPheGly                              160165170                                                                     IleProCysThrAspLysPheValValPheSerGlnIleGluIleLeu                              175180185                                                                     ThrProProAsnMetThrAlaLysCysAsnLysThrHisSerPheMet                              190195200205                                                                  HisTrpLysMetArgSerHisPheAsnArgLysPheArgTyrGluLeu                              210215220                                                                     GlnIleGlnLysArgMetGlnProValIleThrGluGlnValArgAsp                              225230235                                                                     ArgThrSerPheGlnLeuLeuAsnProGlyThrTyrThrValGlnIle                              240245250                                                                     ArgAlaArgGluArgValTyrGluPheLeuSerAlaTrpSerThrPro                              255260265                                                                     GlnArgPheGluCysAspGlnGluGluGlyAlaAsnThrArgAlaTrp                              270275280285                                                                  ArgThrSerLeuLeuIleAlaLeuGlyThrLeuLeuAlaLeuValCys                              290295300                                                                     ValPheValIleCysArgArgTyrLeuValMetGlnArgLeuPhePro                              305310315                                                                     ArgIleProHisMetLysAspProIleGlyAspSerPheGlnAsnAsp                              320325330                                                                     LysLeuValValTrpGluAlaGlyLysAlaGlyLeuGluGluCysLeu                              335340345                                                                     ValThrGluValGlnValValGlnLysThr                                                350355                                                                        __________________________________________________________________________

We claim:
 1. Human IL3 receptor α-chain subunit substantially free ofother human proteins.
 2. The α-chain subunit of claim 1 comprising SEQID NO:
 2. 3. The α-chain subunit of claim 1, capable of forming anoperable association with a β-chain of a human interleukin-3 receptor.4. The α-chain subunit of claim 3, wherein said operable associationprovides for binding human IL-3 with a binding constant at least anorder of magnitude less than by either α-chain or β-chain alone.
 5. Theα-chain subunit of claim 1, which is a recombinant protein.
 6. Theα-chain subunit of claim 5, made in E. coli, yeast, or a mammalian cell.7. A method of identifying an IL-3 receptor ligand, said methodcomprising the steps of:expressing a recombinant gene for human IL-3receptor α-chain subunit, and a gene for β-chain ofinterleukin-3-receptor so that said α- and β-chain operably associate toform a high affinity interleukin-3-receptor; contacting said IL-3receptor with a sample comprising a putative ligand for said receptor;and determining whether said putative ligand affects binding of IL-3 tosaid high affinity receptor for IL-3.
 8. The method of claim 7, whereinsaid ligand is an agonist or antagonist of said receptor.
 9. The methodof claim 7 wherein said expressing is in a cell.
 10. The method of claim9 wherein said cell is stably transformed with a first vector carryingsaid gene for α-chain and a second vector carrying said gene forβ-chain.
 11. The method of claim 10 wherein said first vector is pDUK-1,said second vector is pKH97 and said high affinityinterleukin-3-receptor has a binding constant with human interleukin-3of less than 1 nM.
 12. The method of claim 9, wherein said cell is E.coli, yeast, or a mammalian cell.
 13. The method of claim 7, whereinsaid α-chain comprises SEQ ID NO:
 2. 14. The method of claim 13, whereinsaid gone comprises SEQ ID NO:1.
 15. A composition of matter comprisinga recombinant human IL-3 receptor α-subunit and a β-chain of aninterleukin-3-receptor, said α-chain subunit and β-chain being inoperable association.
 16. The composition of matter of claim 15 whereinsaid α-chain subunit is encoded by the cDNA insert of pDUK-1 and saidβ-chain is encoded by the cDNA insert of pKH97.
 17. The composition ofclaim 5, wherein said composition binds human interleukin-3 with abinding constant at least an order of magnitude less than by eitherα-chain or β-chain alone.
 18. The composition of claim 17, wherein saidcomposition provides a binding constant of less than 1 nM.
 19. Thecomposition of claim 18, wherein said binding constant is less than 200pM.
 20. The α-chain subunit of claim 15, comprising SEQ ID NO:
 2. 21.The α-chain subunit of claim 15, wherein said subunit is a recombinantprotein.
 22. The α-chain subunit of claim 21, made in E. coli, yeast, ora mammalian cell.
 23. A cell comprising a composition of claim
 15. 24.The cell of claim 23, wherein said operable association confers IL-3responsiveness onto said cell.
 25. A recombinant polypeptide comprisingSEQ ID NO:
 2. 26. The polypeptide of claim 25, made by expressing anucleic acid comprising SEQ ID NO:
 1. 27. A cell comprising apolypeptide of claim
 26. 28. The cell of claim 27, which is E. coli,yeast, or a mammalian cell.
 29. A method of determining the effect on anIL-3 receptor by a reagent, said method comprising the stepsof:combining a polypeptide of claim 25 with a β-chain of aninterleukin-3-receptor so that said α- and β-chain gene productsoperably associate to form a high affinity interleukin-3-receptor; andmeasuring the effect of said reagent on said high affinity receptor forIL-3.